348 
Fishery Bulletin 111(4) 
Table 3 
The ratio of the number of S-phase nuclei to the number of Gl-phase nuclei with high 
nuclear RNA content (RSG1) and values of nuclear RNA (nRNA) fluorescence for larvae 
of Walleye Pollock ( Gadus chalcogrammus ) sampled from always-fed and unfed treat- 
ments reared at different temperatures in 2010. Fluorescence values are arbitrary units 
and were adjusted on the basis of controls to make samples comparable among sessions 
of flow cytometry. Standard errors of geometric means of nRNA fluorescence (all cell- 
cycle phases pooled) and of RSG1 means are reported in parentheses. Cell-cycle phases: 
Gl=gap 1 or cell growth before cell division, G0=resting state from G1 phase, S=DNA 
synthesis, G2=gap 2 or cell growth before mitosis, and M=mitosis. 
Rearing temperature (°C) 
Treatment 
n 
RSG1 
nRNA 
fluorescence 
2.9 
Unfed 
28 
0.14 (0.01) 
32.3 (1.5) 
2.9 
Always-fed 
26 
0.26(0.02) 
32.5 (2. OF 
5.9 
Unfed 
13 
0.09(0.01) 
26.7 (0.8) 
5.9 
Always-fed 
24 
0.19(0.01) 
30.5 (1.9) 2 
8.7 
Unfed 
17 
0.13(0.02) 
26.5 (1.1) 
2 n= 14. 
2 n= 10. 
the sizes of healthy and unhealthy larvae overlap in 
that size class. 
Nuclear RNA varied with rearing temperature, in- 
creasing as temperature decreased, a result similar to 
the findings of other studies on the effect of temper- 
ature on RNA content of larval fishes (Canino, 1994; 
Malzahn et al., 2003). This result also indicates that 
our nRNA staining protocol worked as intended. 
Temperature affected RSG1, and, therefore, it needs 
to be accounted for when our method is used to mea- 
sure the condition of larvae sampled from the field. 
RSG1 for healthy larvae was smaller at warmer tem- 
peratures (5.9°C in our study), and this observation 
may indicate that nuclei were cycling faster through 
the cell cycle than nuclei at colder temperatures. Oth- 
er studies have shown that increasing temperature 
decreases cell-cycle duration in other species, such as 
yeast ( Saccharomyces cereuisiae) (Jagadish and Carter, 
1978) and Magellan Plunderfish (Harpagifer bispinis) 
(Brodeur et al., 2003). There was no difference in the 
percentage of S-phase brain cells of fed larvae of At- 
lantic Cod (Gadus morhua) reared at 6°C and 10°C — a 
finding that was explained by an increased rate of pro- 
gression by cells through the cell cycle at the higher 
temperature (Gonzalez-Quiros et al., 2007). A similar 
result was also found for S-phase nuclei from muscle 
cells of larvae of Walleye Pollock reared at 3.2°C and 
5.9°C, temperatures comparable to those used in our 
study (Porter and Bailey, 2011). 
RSG1 of unhealthy larvae was not affected by tem- 
perature, probably as a result of the slow or ceased 
growth of these larvae. The brain cells of starved At- 
lantic Cod larvae reared at 10°C had a smaller per- 
centage of S-phase cells than the brain cells of larvae 
starved at 6°C (Gonzalez-Quiros et al., 2007), a differ- 
ence that may be due to the length of time that the 
larvae were starved. Fish larvae in general starve 
faster at higher temperatures, and, for larvae of Wall- 
eye Pollock in a previous study, the percentage of S- 
phase nuclei decreased the longer larvae were starved 
(Porter and Bailey, 2011). Atlantic Cod larvae at both 
temperatures were starved for 5 days; therefore, the 
percentage of S-phase cells of the larvae starved at the 
higher temperature (10°C) would be expected to be less 
than the percentage for the larvae starved at the lower 
temperature. 
The loss of DNA during freezing and thawing has 
been documented for human blood, where about 25% 
of the DNA was lost (Ross et al., 1990). Differences 
in nuclear membrane permeability among cell-cycle 
phases may account for the loss of DNA when larvae 
of Walleye Pollock were frozen and thawed, and they 
may explain why there was a decrease in the percent- 
age of muscle cell nuclei in the S phase. S-phase nuclei 
may be more permeable than G2-phase nuclei (Coverly 
et al., 1993; Leno and Munshi, 1994), resulting in diffu- 
sion of DNA out of the nucleus, and they may rupture 
because they may be more fragile than nuclei at other 
phases — an outcome that also would contribute to loss 
of nuclei. Crytoprotectant has been used to stabilize 
DNA in brain cells of Walleye Pollock larvae during 
freezing (Theilacker and Shen, 1993a), and it could 
possibly be used to prevent the loss of DNA from mus- 
cle cell nuclei as well, but the use of cryptoprotectant 
needs further investigation, particularly for between- 
laboratory comparisons where standardized protocols 
are used (Caldarone et al., 2006). In our study, the loss 
of DNA was unchanged for up to 10 months when the 
